Lyme disease is a zoonosis caused by the tick-borne spirochaete B. burgdorferi. When a susceptible host is bitten by an ixodid tick, B. burgdorferi organisms enter the skin. In humans the initial skin manifestation is termed erythema chronicum migrans (ECM) whereas a long-standing infection of the skin produces acrodermatitis chronica atrophicans. The Borrelia organisms also enter the circulatory system of the host and are distributed to various organs, including the brain and joints. A secondary spread of the pathogens produces a variety of clinical syndromes, including lymphocytic meningoradiculitis, myocarditis and chronic arthritis. In many patients the infection of some tissues, particularly the brain and joints, persists for years and can be severely disabling. These forms of chronic Lyme disease are a consequence of the host's inability to rid itself of the infectious agent and perhaps the development of an autoimmune reaction.
Diagnosis of Lyme disease has chiefly been based on clinical evidence. The best marker during the primary stage of infection has conventionally been the presence of erythema chronicum migrans (ECM) but these skin lesions may not always develop or they may manifest atypically. Moreover, Lyme disease can be confused with other illnesses characterized by neurologic or arthritic manifestations. When clinical histories are incomplete, serologic testing with determination of antibody titers is the best conventionally laboratory method of diagnosis. Indirect fluorescent antibody (IFC) staining tests and enzyme-linked immunosorbent assays (ELISA) are used to detect total immunoglobulins or class-specific IgM and IgG antibodies to B. burgdorferi. ELISA is usually preferred because the procedures are more easily standardized and automated and because absorbance values can be statistically analyzed to give more objective results.
B. burgdorferi spirochaetes are helically shaped, motile cells with an outer cell membrane that surrounds a protoplasmic cylinder complex, consisting of the cytoplasm, the cell wall, the inner cell membrane and the flagella which are located not at the cell surface but in the periplasmic space between the outer cell membrane and the protoplasmic cylinder. The outer cell membrane and the flagella are assumed to play an important role in the host-parasite interactions during the disease and has been subjected to several investigations, identifying major surface-exposed proteins as important immunogens.
It has been shown that the earliest IgM antibodies formed against antigens of the B. burgdorferi strain B31, which was deposited in the American Type Culture Collection in 1983 with the accession number ATCC 35210, are directed against a genus-specific flagellar polypeptide termed flagellin having a molecular weight of 41 kd (18) and which reacts with monoclonal antibody H9724. IgG antibodies are also first directed to the 41 kg flagellin, but with advancing disease IgG antibodies form against other immunogens, especially against two abundant proteins with molecular weights of 31 kg and 34 kd. These two proteins, which have been denoted OspA (31 kd) and OspB (34 kd), have been found to be located at the B. burgdorferi surface and embedded in its outer fluid cell membrane. The OspA protein has been found to be less variable in its molecular weight and in its reactivity with monoclonal antibody H5332 (10), whereas the molecular weight of OspB proteins from different B. burgdorferi strains vary and the OspB proteins of different strains also show varying reactivity with two monoclonal antibodies against OspB (H6831 and H5TS) (9). The main variation among OspA proteins is found between isolates from Europe and the United States.
Conventional diagnostic tests for Lyme disease have used whole spirochaetal sonic extracts as test antigens in ELISA to detect antibodies to B. burgdorferi, but this test yields unsatisfactory low diagnostic sensitivity (20 to 60%) during the early stage of infection, possibly due to a slow and late-appearing antibody response and to the inclusion of irrelevant cross-reacting antigens in the whole-cell preparations. In addition, the use of whole cells as test antigens may result in the occurrence of false positive reactions. For example, among patients with syphilis and in areas where a closely related relapsing fever Borrelia spp. co-exist with B. burgdorferi, serologic differentiation of Lyme disease from tick-borne relapsing fever is difficult. Detection of IgG antibody to B. burgdorferi in later stages of infection can help in distinguishing Lyme disease form aseptic meningitis, multiple sclerosis, serum negative rheumatoid arthritis, juvenile rheumatoid arthritis, and Reiter's syndrome.
The antigen-antibody diagnostic approach has been found not to work as well in connection with Lyme disease as in connection with many other infectious diseases.
One of the reasons for this is that only a low number of spirochaetes is present and that the antigens in the outer membrane of the spirochaetes are hard to detect for the immune system of the infected organism.
Another reason is that the antibody response to the B. burgdorferi infection first arises weeks after the bite of the tick, and in many cases first after the patient has shown clinical signs of the disease.
It would be desirable to provide a diagnostic tool which is able to diagnose a B. burgdorferi infection at all stages, also, at very early stages even before the clinical signs of infections appear and the diagnostic tool being independent of the causative infective B. burgdorferi strain.
A most promising and sensitive method for diagnosing the Lyme disease agent is that of detecting a single B. burgdorferi organism by PCR amplification.
Nielsen S. L. et al. Molecular and Cellular Probes (1990) 4, 73-79, Detection of Borrelia burgdorferi DNA by the polymerase chain reaction, and Malloy, D. C. et al, Journal of Clinical Microbiology, June 1990, p. 1089-1093, Detection of Borrelia burgdorferi Using the Polymerase Chain Reaction, both disclose the use of DNA-sequences from only B. burgdorferi strain B31 in the preparation of primers useful in the PCR-DNA diagnostic of Lyme disease.
WO91/06676 discloses the use of DNA primers associated with the SC plasmid in the diagnostic of Lyme disease, but does not disclose the sequence of the primers.
ERP 421 725 A1 discloses nucleic acid probes useful for identifying B. burgdorferi in samples. The probes are designed from B. burgdorferi strains in The United States and Europe.
EP 445 135 discloses the use of a DNA fragment from the OspA gene from the B31 strain in PCR-DNA diagnostic of B. burgdorferi infection in mammals, including humans, as well as the use of immunogenic polypeptides found to be antigenic when assessed with monoclonal antibodies directed against OspA in the preparation of a vaccine immunizing mammals, including humans, against Lyme disease. EP 445 135 discloses, inter alia, three contemplated epitopes which are small fragments of B31 OspA: Lys-Glu-Lys-Asn-Lys-Asp, Ser-Lys--Lys-Thr-Lys-Asp, and Lys-Ala-Asp-Lys-Ser-Lys. To the extent this is relevant, these fragments and their utility and use as epitopes is disclaimed in the present invention.
Rosa, P. A. et al., Journal of Clinical Microbiology, March 1991, p.524-532, Polymerase Chain Reaction Analyses Identify Two Distinct Classes of Borrelia burgdorferi discloses PCR primers used in the identification.
One object of the present invention is to provide non-immunological assays by providing nucleotide sequences which can hybridize with different strains of B. burgdorferi from different geographical regions so that a diagnostic tool for detecting B. burgdorferi spirochaetes independent of causative infective strain of B. burgdorferi is obtained. Another object is to provide novel DNA fragments related to B. burgdorferi. A further object of the invention is to provide antigenic polypeptides, an antigenic composition and a vaccine for immunizing animals, including humans, against Lyme disease substantially independent of the infective B. burgdorferi strain causing the disease.